The increasing use of recombinant DNA technology to produce transgenic plants for commercial and industrial use requires the development of high-throughput methods of analyzing transgenic plant lines. Such methods are needed to maintain transgenic plant varieties through successive generations, to prevent the escape of transgenes into the environment, and to assist in the rapid development of transgenic plants with desirable or optimal phenotypes. Moreover, current guidelines for the safety assessment of GM plants proposed for human consumption requires characterization at the DNA and protein level between the parent and transformed crop. Sesikeran and Vasanthi (2008) Asia Pac. J. Clin. Nutr. 17 Suppl. 1:241-44. New plant varieties that are developed consist of increasingly complex genetic modifications including, inter alia, stacked genes and traits.
The current methods for analysis of transgenic plants that are preferred in the art are: DNA-based techniques (e.g. PCR); RT-PCR; the use of reporter genes; Southern blotting; and immunochemistry. All of these methodologies suffer from various shortcomings, and a superior method that is broadly able to rapidly and inexpensively identify and quantitate multiple transgenic gene products in a high-throughput manner from a limited sample from a transgenic plant is desired.
DNA-based techniques for transgenic plant analysis suffer from several notable deficiencies. Despite the fact that Agrobacterium-mediated transformation is the most preferred method of genetic plant transformation, the genotypes of Agrobacterium-transformed plants are difficult to analyze by PCR-based methodologies. See Nain et al. (2005) Plant Mol. Biol. Rep. 23:59-65. The presence of even trace amounts of Agrobacterium in transformed tissues yields misleading PCR results. Id. DNA amplification formats also require empirical testing of gene-specific primers and thermocycler conditions. Most significantly, DNA-based approaches for screening transgenic plants do not actually determine expression of the gene product protein. Similarly, RT-PCR or Northern blot analysis may be used to confirm the presence of transgene transcripts in transgenic plant material. Alwine et al. (1977) Proc. Nat. Acad. Sci. 74:5350-54; Toplak et al. (2004) Plant Mol. Biol. Rep. 22:237-50. Neither do these methods confirm the presence of actual protein expression in the source plant material. These techniques also require the use of radioactive materials and/or large amounts of tissue and processing time.
Reporter genes, such as genes encoding fluorescent proteins, may also be co-transformed into transgenic plants to provide a tool to identify transformants. However, reporter genes are only indirect reporters of genetic recombination. Expression of the reporter gene construct does not confirm expression of the accompanying transgene. Further, either the reporter gene or the transgene may be lost in successive generations of the host plant, thereby uncoupling presence of the reporter from the transgene of interest. Similarly, transgenes may escape from the host plant into neighboring plants, for example by cross-pollination, without concurrent escape of the reporter gene. When multiple genes are stacked in a transgenic plant, an equal number of reporter genes would have to be introduced to analyze the transgenic proteome, and since reporter gene function is only an indirect reporter of transgene function, changes in expression of one transgene in response to the presence of an additional transgene would not be detected.
Unlike the methods outlined above, immunochemistry can be used to identify products of transgene expression in a transgenic plant. Though immunochemistry is useful for this purpose, the method requires highly-purified protein samples for antibody production. The resulting antibodies must be tested for specificity, and reagent-specific assay conditions must be developed. The high levels of expression and purification required to conduct immunochemistry, as well as the related problem of removing contaminants from the plant tissue, are limitations on the utility of this method.
Mass spectrometry may also be used to analyze the proteome of a transgenic plant. However, art-recognized spectrometric techniques require complex mixtures of plant proteins to be first separated by 2-D gel electrophoresis. Rajagopal and Ahern (2001) Science 294(5551):2571-73; See also Domon and Aebersold (2006) Science 312(5771):212-17, 214. Single bands from the gel-separated protein sample may then be digested with a protease and subjected to mass spectrometry to identify the unique protein originally present in the undigested band. See, e.g., Chang et al. (2000) Plant Physiol. 122(2):295-317. The gel-separation step in this method is a time-consuming process that impedes the use of mass spectrometry in high-throughput applications.
There is a need in the art for a high-throughput method for detecting and quantitating the presence of products of transgene expression in plants that does not require purified or highly-expressed protein, or method-specific reagents. This method will be useful in helping cultivators and growers of transgenic plants maintain the phenotype of the target transgenic plant variety through successive generations of sexual and/or asexual reproduction. The method will also be useful in rapidly analyzing product plants of a transformation procedure to identify those product plants that are transgenic plants and express the introduced protein in desired tissues. Further, the method may be used to rapidly screen plants at risk of being contaminated with transgenes from a transgenic plant, in order to accomplish bioconfinement of the transgenic plant.